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20 March 2019, Volume 46 Issue 3
Biotechnology
Cloning,Expression and Bioinformatics Analysis of Atg12 Gene in Apis cerana cerana and Apis mellifera
TU Yangyang, WU Jiangli, WU Pengjie, TAN Jing, YU Huimin, XU Jin, GUO Yueqin, WANG Lihua, XU Shufa
2019, 46(3):  643-651.  doi:10.16431/j.cnki.1671-7236.2019.03.001
Abstract ( 229 )   PDF (2026KB) ( 286 )  
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The purpose of this study was to investigate the expression differences of autophagy-related 12 (Atg12) gene in Apis cerana cerana and Apis mellifera,and provide references for studying the mechanism of Apis cerana cerana resitance to Varroa destructor.The primers were designed according to the Atg12 gene sequence of Apis cerana in GenBank (accession No.:XM_017048509.1).The Atg12 gene was amplified from the head of Apis cerana cerana and Apis mellifera,then was cloned,sequenced and expressed prokaryoticly,and the amino acid sequence and protein structure were analyzed.Meanwhile,Real-time PCR was used to analyze the different expression of Atg12 gene in head tissues of Apis cerana cerana and Apis mellifera.The results showed that the target fragment size was 450 bp and the recombinant protein size was about 42 ku.Genetic phylogenetic tree analysis showed that the relationship among Apis cerana cerana,Apis mellifera,Apis dorsata and Apis florea was closer in the evolution of Atg12 gene.Bioinformatics analysis of the recombinant protein showed that the secondary structure of the protein contained 6 polypeptide binding sites,6 β-sheets and 4 α-helices,the molecular weight was about 16.13 ku,and the isoelectric point was 6.73.The results of Real-time PCR showed that the expression of Atg12 gene in the head tissue of Apis cerana cerana was extremely significantly higher than that of Apis mellifera.The results of this study provided references for further study on the mechanism of action of Atg12 gene in Apis cerana cerana resitance to Varroa destructor.

Overexpression and Interference of FADS2 Gene in Mammary Gland Cells of Dairy Cows
MA Xiaoya, PANG Chunying, DENG Tingxian, DUAN Anqin, LU Xingrong, LIANG Shasha, LIANG Xianwei
2019, 46(3):  652-660.  doi:10.16431/j.cnki.1671-7236.2019.03.002
Abstract ( 201 )   PDF (2610KB) ( 149 )  
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In order to explore the role of fatty acid desaturases 2 (FADS2) gene in fatty acid metabolism in dairy mammary gland cells,this study overexpressed and interfered with FADS2 gene in dairy mammary epithelial cells,and studied the regulation of FADS2 gene on fatty acid synthesis related genes and the effect on triglyceride content in dairy mammary epithelial cells.The siRNA and overexpression vector pcDNA3.1-FADS2-EGFP were designed according to the CDS sequence of FADS2 gene.The effect of FADS2 gene overexpression and interference on the expression of fatty acid metabolism related genes and the change of triglyceride content in cells were detected by transfecting mammary gland cells.The results showed that the overexpression vector pcDNA3.1-FADS2-EGFP and the interference fragment were successfully obtained,and the transfected cells had good overexpression and interference effects.After overexpression of FADS2 gene,AGPAT1,SCAP,GPAM,ELOVL5,ACAA1,FADS1,DGAT1 and PPARα genes were significantly down-regulated (P<0.05),and PLIN2 gene was extremely significantly up-regulated (P<0.01).After FADS2 gene interference,AGPAT1,GPAM,ELOVL5,ACAA1,PLIN2 and FADS1 genes were significantly up-regulated (P<0.05),INSIG1 gene was extremely significantly up-regulated (P<0.01),andDGAT1 and PPARα genes were significantly down-regulated (P<0.05).Triglyceride assay showed that FADS2 gene overexpression and interference could reduce the content of triglyceride in mammary gland epithelial cells.In summary,FADS2 gene could regulate the expression of lipid synthesis-related genes in mammary gland epithelial cells,and had a regulatory effect on breast lipid synthesis.

Cloning,Prokaryotic Expression and Bioinformatics Analysis of recN Gene of Pasteurella multocida
AZHANG Luyin, ZHENG Yiying, WANG Chengqiang, AN Qi, ZHANG Mengmeng, HUAGN Haifeng, ZHANG Zhenxing, LI Baobao, ZHU Shu, YANG Xiaojian, CAO Ruiyong, NIE Xin, DU Li, WANG Fengyang
2019, 46(3):  661-668.  doi:10.16431/j.cnki.1671-7236.2019.03.003
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This experiment was aimed to study the clone and prokaryotic expression of recN gene of Pasteurella multocida,and analyze its protein by bioinformatics method.A pair of primers was designed according to the recN gene sequence in GenBank(accession No.:CP003313.1),and the target gene fragment was obtained by PCR.The recombinant plasmid pET-28a(+)-recN was constructed and transformed into E.coli DH5α competent cells,and the plasmid was extracted for restriction enzyme digestion.The correct recombinant plasmid was transformed intoE.coli BL21(DE3) competent cells.The fusion protein was induced by IPTG,and identified by SDS-PAGE and Western blotting.The results showed that the recN gene with a size of about 1 677 bp was successfully cloned in this experiment.The size of the His-Tag fusion protein induced by expression was about 66.94 ku,and it mainly existed in the form of inclusion body.According to bioinformatics analysis,the recN protein had the molecular formula C2735H4428N786O855S16,the extinction coefficient was 24 785,the instability coefficient was 43.99,which was unstable protein;The theoretical isoelectric point (pI) was 5.62,which was acidic protein;The total average hydrophilicity was -0.316,which was the same hydrophobic protein as the experimentally expressed inclusion body protein;The half-life of reticulocytes in mammalian was estimated to be 30 h,the half-life in yeast and E.coli (in vivo) was more than 20 and 10 h,respectively.The secondary structure prediction was mainly alpha helix (64.87%) and random coil (21.00%);The hydrophobic analysis results showed that recN protein had 3 highly hydrophobic sexual regions and 9 highly hydrophilic regions.This results provided a reference basis for further exploration of the function of recNgene of Pasteurella multocida.

Animal Nutrition and Feed Science
Effects of Bacillus coagulans on Intestinal Function in Weaned Piglets
WU Mengjun, JI Changzheng, LI Siyuan, DONG Yi, ZHAO Guangyu, ZHANG Yue, WANG Lei, ZHAO Di, YI Dan, HOU Yongqing
2019, 46(3):  669-676.  doi:10.16431/j.cnki.1671-7236.2019.03.004
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The study was to investigate the effects of Bacillus coagulans (BC) on intestinal function in weaned piglets.Twenty-four piglets were randomly allocated into three groups.The control group was fed the based diet,whereas the BC Ⅰ and BC Ⅱ groups were fed the basal diet supplemented with 2×106 and 2×107 CFU/g Bacillus coagulans,respectively.On the 21th day of the trial,D-xylose (0.1 g/(kg·BW)) was orally administrated to all piglets.One hour later,jugular vein blood samples were collected,and then all pigs were sacrificed to obtain the mid-ileum and the chyme of ileum and colon.The results showed that,cmpared with the control group,plasma diamine oxidase (DAO) activities in BC Ⅰ and BC Ⅱ groups were significantly decreased (P<0.05),and plasma D-xylose content was significantly increased in BC Ⅰ group (P<0.05).The actitivies of ileum glutathione peroxidase (GSH-Px) in BC Ⅰ and BC Ⅱ groups increased significantly (P<0.05),and the content of ileal glutathione (GSH) in BC Ⅱ group increased (P>0.05).The content of ileal malondialdehyde (MDA) was significantly decreased (P<0.05);The mRNA expression of ileal mucosa aquaporin 3 (AQP3),basic amino acid transporter (b0,+AT) and sodium glucose cotransporter 1 (SGLT-1) in BC Ⅰ group increased significantly (P<0.05).The mRNA expression levels of ileum mucosa AQP3,AQP10,inward rectifier potassium channel 13 (KCNJ13),b0,+AT,SGLT-1 were significantly increased in BC Ⅱ group (P<0.05).The number of Enterococcus,Bifidobacterium in the ileum and colon of BC Ⅰ group were significantly increased (P<0.05).The number of Enterococcus in the ileum,Enterococcus,Bifidobacterium in colon of BC Ⅱ group were significantly increased (P<0.05).In summary,the addition of Bacillus coagulans in the diet could improve the intestinal absorption,transport capacity and antioxidant capacity of weaned piglets,and improve the intestinal flora structure of weaned piglets.

Study on Antioxidant and Anti-inflammatory Effects of Extracts from Piper sarmentosum Roxb. in vitro
CHEN Chuanwei, ZHOU Luli, WANG Dingfa, ZHOU Hanlin
2019, 46(3):  677-683.  doi:10.16431/j.cnki.1671-7236.2019.03.005
Abstract ( 274 )   PDF (711KB) ( 98 )  
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This study was aimed to investigate the active ingredient and evaluate the antioxidant and anti-inflammatory activities of Piper sarmentosum Roxb. extracts in vitro.The antioxidant activities of three pepper extracts (petroleum ether,n-butanol and ethyl acetate) in vitro were determined by ABTS method.The inflammation model of IPEC-J2 cell was established with LPS in vitro,the levels of tumor necrosis factor (TNF-α),interleukin-1 (IL-1) and interleukin-6 (IL-6) were detected in cell supernatant by ELISA after pretreatment of Piper sarmentosum Roxb. extract.The results showed that the total antioxidant activity of Piper sarmentosum Roxb. extract in descending order were n-butanol extract,ethyl acetate extract and petroleum ether extract (P<0.05).The contents of total alkaloids and total phenols of Piper sarmentosum Roxb. n-butanol extract were the highest,which were 2.81 mEq/100 g and 161.82 mg/g,respectively.In anti-inflammatory experiments,compared with the blank group,the levels of TNF-α(P<0.05),IL-1(P>0.05) and IL-6 (P<0.05) in IPEC-J2 cells increased in LPS group,while compared with the Piper sarmentosum Roxb. pretreatment groups,these indexes in LPS group decreased significantly (P<0.05).The concentrations of cytokines from high to low were ethyl acetate extract,petroleum ether extract and n-butanol extract.In conclusion,the extract of Piper sarmentosum Roxb. had antioxidant activity,and it could affect the secretion of cytokines in IPEC-J2 cells and inhibit the inflammation.

Effect of Compound Plant Essential Oils on Structure of Intestinal Mucosa and Antioxidant Capacity of Weaned Piglets Challenged with Lipopolysaccharide
SONG Zhuan, ZHAO Guangyu, DONG Yi, JI Changzheng, LI Siyuan, WANG Lei, YI Dan, HOU Yongqing
2019, 46(3):  684-689.  doi:10.16431/j.cnki.1671-7236.2019.03.006
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In order to investigate the effect of compound plant essential oil (OCT) on structure of intestinal mucosa and antioxidant capacity of weaned piglets challenged with lipopolysaccharid (LPS),twenty-four weaned piglets (28-day-old,Duroc×Landrace×Yorkshire) were randomly assigned into 3 groups:Control group,LPS group and OCT group.Piglets in the control and LPS groups were fed a basal diet,and piglets in the OCT group received the basal diet+50 mg/kg OCT.On the 21th day of the trail,piglets in the LPS and OCT groups were injected intraperitoneally with LPS (100 μg/(kg·BW)),and piglets in the control group were injected with the same volume of physiological saline.Venous blood samples were obtained at 3 h post LPS or saline injection,all piglets were killed to obtain the intestinal mucosa for analysis at 6 h post LPS or saline injection.Compared with the control group,LPS stimulation had a tendency to decrease villus height/crypt depth in jejunal mucosa (P>0.05),it could cause a significant reduction in villus height and villous height/crypt depth in ileal mucosa;the activity of CAT and SOD in plasma,and SOD in jejunum mucosa were decreased significantly (P<0.05),it also increased the activity of iNOS in jejunum mucosa,the content of H2O2 in plasma and ileum mucosa significantly(P<0.05).Compared with the LPS group,adding OCT could increase villus height and villus height/crypt depth in jejunal mucosa,villus height in ileal mucosa significantly (P<0.05),and villus height/crypt depth in ileal mucosa had a tendency to increase (P>0.05);The activity of CAT in plasma and SOD in jejunum mucosa were also increased significantly (P<0.05),the content of H2O2 in plasma and ileum mucosa were decreased significantly (P<0.05).In conclusion,the addition of 50 mg/kg OCT to the diet could alleviate the oxidative stress induced by LPS challenge to a certain extent and improve the intestinal structure of piglets.

Effect of Supplemented with Rumen-protected 5-hydroxytryptophan on Melatonin Content and Microbial Communities in Intestinal Tract Digesta of Sheep
ZHAO Fang, WANG Gen, ZHAO Guodong, GAO Chao, LI Xiaobin, MA Chen, YANG Kailun
2019, 46(3):  690-702.  doi:10.16431/j.cnki.1671-7236.2019.03.007
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This study was aimed to investigate the effects of rumen protected 5-hydroxytryptophan (RPT-5-HTP) supplementation on melatonin content and microbial communities in gastrointestinal tract digesta of sheep,and explored the possibility of regulating sheep gastrointestinal tract microbial communities by RPT-5-HTP.Fifteen Kazak sheep which were 3 year old,average body weight (47.79±3.70) kg were chosen and divided into 3 groups:Control group,groups Ⅰ and Ⅱ.All sheep were fed with 12 g/(kg·BW) powder concentrate,1.8 kg corn silage every day,and had free continuous access to mixed hay.Additionally,each sheep in groups Ⅰ and Ⅱ was fed with RPT-5-HTP 111,222 mg/(kg·BW),respectively.The supplementary feeding experiment lasted for 25 d.At the end of the experiment,all sheep were slaughtered after the morning feeding (6 h),and the contents of jejunal,ileal,colonic and cecal digesta were taken to determine melatonin content and microflora structure.The results showed that the melatonin concentration in colonic digesta content of groups Ⅰ and jejunal and colonic digesta of groups Ⅱ were extremely significantly higher than that in control group (P<0.01),and that in ileal and cecal digesta of groups Ⅰ,Ⅱ were lower than control group,but there was no significant differences among three groups (P>0.05).The ACE and Chaol indexes of bacterial flora in the jejunal,cecal digesta of groups Ⅰ,Ⅱ were lower than those in control group (P>0.05).Compared with the control group,the bacterial diversity in cecal and colonic digesta were not significantly different at the levels of phylum and genus (P>0.05).The relative abundance of Firmicutes in the jejunal digesta was significantly increased in group Ⅰ (P<0.05),and the relative abundance of Tenericutes in group Ⅰ was significantly lower than that in control group (P<0.05).At the genus level,the relative abundance of Aeriscardovia in the jejunal digesta of group Ⅱ was increased (P<0.05).The relative abundance of Ruminococcaceae-NK4A214 in ileal digesta of groups Ⅰ,Ⅱ were significantly increased (P<0.05),while relative abundance of Clostridium-sensu-stricto and Romboutsia were significantly decreased (P<0.05).In conlusion,the RPT-5-HTP could increase melatonin concentration in the colonic digesta,but had no significant effect on the bacterial diversity of the gastrointestinal tract.

Effect of Fermented Fruit Pomace in Diet on Growth Performance and Apparent Digestibility of Nutrient in Duhua Binary Finishing Pigs
ZHANG Ziwei, CHEN Yueli, LU Huayan, LIANG Yingdan, CHEN Cuihuai, YANG Linfang
2019, 46(3):  703-710.  doi:10.16431/j.cnki.1671-7236.2019.03.008
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The purpose of this experiment was to study the effects of fermented pomace on the growth performance,apparent digestibility of nutrients and the duration of feed through the digestive tract.A total of 480 Duhua binary breeding pigs (♂Duroc pig×♀Guangdong small-eared pigs) with similar body conditions were selected and randomly divided into experimental group and control group,24 replicates per group,10 pigs per replicate.The control group was fed with the basal diet,and the experimental group was fed with the basal diet,and 5% to 20% of the fermented pomace was added according to the growth stages of the pig.The feeding trial lasted for 5 months.The results showed that in the piglet stage (25 to 60 kg stage,adding 5% to 10% fermented pomace),the average daily gain did not affect,the test group's full-price feed intake decreased by 6.12% (P>0.05),and the F/G decreased 5.57% (P>0.05).In the middle pig stage (60 to 80 kg stage,adding 15% fermented pomace),the average daily weight gain of the test group increased by 6.50% (P>0.05),the full-price feed intake decreased by 7.55% (P>0.05),and the F/G decreased by 12.90% (P<0.05).In the large pig stage (80 to 100 kg stage,adding 20% fermented pomace),the average daily weight gain of the test group increased by 4.69% (P>0.05),the full-price feed intake decreased by 9.50% (P>0.05),and the F/G decreased by 12.20% (P<0.05).During the whole trial period,the average daily gain of the experimental group increased by 2.08% (P>0.05),the feed intake of the full-price feed decreased by 7.47% (P>0.05),and the F/G decreased by 8.62% (P<0.05).The addition of fermented pomace could significantly improve the apparent digestibility of protein in the middle and late stages of Duhua binary fattening pigs,but the absorption of calcium and phosphorus had a decreasing trend.And it could shorten the time of the feed through the digestive tract of the Duhua binary fattening pigs in initial stage (P>0.05),but could extend the time of the feed through the digestive tract in medium and late stages (P>0.05).The results of this experiment showed that adding an appropriate amount of fermented pomace in the diet of Duhua binary fattening pigs could improve the growth performance,reduce the F/G,and to some extent,prolong the retention of feed in the digestive tract of the pig in the middle and late fattening period and promote the digestion and absorption of nutrients such as CP in the diet.

Screening and Identification of A Cellulase-producing Bacterium and Analysis of Its Cellulase Characterization
LI Hao, BAI Guangjian, LAN Nan, XU Jing, ZHAO Xingxiu, ZOU Wei
2019, 46(3):  711-718.  doi:10.16431/j.cnki.1671-7236.2019.03.009
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In order to isolate cellulose-producing strain from soil samples of decayed leaf,the screening and identification of a cellulase-producing bacterium and its cellulase characterization were measured in this study.Firstly,the cellulose-producing strain was preliminarily isolated by carboxymethyl cellulose sodium (CMA-Na) culture medium,and then a high-yield cellulase strain B17 was obtained by enzymatic rescreening.Based on morphological observation and 16S rDNA sequencing,B17 was identified as Bacillus subtilis,and then it's fermentation conditions and enzymatic properties were preliminarily explored.The results showed that the optimal fermentation conditions for the cellulose-producting strain B17 were that the cultivate time,temperature and pH were 3 d,35℃ and 6.0.And under this condition,the maximum CMC activity and FPA activity were 85.48 and 59.85 U/mL,respectively.According to the results of enzymatic properties,the optimal temperature and pH for the enzyme were 50℃ and 6.0. Enzyme stability studies showed that enzyme kept active when the temperature was 30 to 60℃ and pH ranged from 4.0 to 7.0.In conclusion,a Bacillus subtilis with high cellulose activity had been isolated and identified,and it had high value for the feed's production of crop straw.

Effects of Different Supplemental Doses of Paraformaldehyde on the Microorganism Quantity and Activities of Digestive Enzymes in the Rumen of Sheep
WANG Xiaodong, LUO Qiujiang, ZANG Changjiang, XIE Jiaxun, CAI Juan
2019, 46(3):  719-731.  doi:10.16431/j.cnki.1671-7236.2019.03.010
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Five male sheep with the body weight of (55.6±3.2) kg,2 to 3 years old,fitted with permanent rumen fistula and 5×5 Latin square design were used in this experiment to study the effects of different supplemental doses of paraformaldehyde on the microbiocoenosis and activities of digestive enzymes in rumen of sheep.The sheep was fed with the same base diet supplemented with 0,150,300,450 and 600 mg/kg paraformaldehyde,respectively.The pH and concentrations of the ammonia nitrogen and volatile fatty acids,the activities of the endocellulase,exocellulase,xylanase,pectinase,cellobiase,amylase and protease activities,the numbers of bacteria and protozoa,DNA copies of fungi in rumen fluid were determined.The results showed that,compared to control group,the groups supplemented with 150,300,450 and 600 mg/kg paraformaldehyde respectively,the voluntary intakes of dry matter were increased by 2.3% (P>0.05),22.1%(P<0.01),7.5% (P<0.01) and -6.6% (P<0.01) respectively;The protozoa numbers in rumen fluid were decreased by 14.2%,62.5%,72.6% and 85.3% (P<0.01) respectively;And the total numbers of bacteria increased by 35.8%,93.8%,-6.8% and -24.2% (P < 0.01),respectively;The copies numbers of fungi were increased by 2.0% (P>0.05),5.4% (P>0.05),14.3% (P<0.01) and 26.8% (P<0.01),respectively;The ammonia N of rumen fluid were decreased by 9.0%,12.0%,14.9% and 20.9% (P<0.01),respectively;The volatile fatty acids were increased by 2.4% (P>0.05),-0.3% (P>0.05),-1.3% (P>0.05) and -10.3% (P<0.01),respectively;The activities of endocellulase were increased by 6.5%,22.4%,-10.2% and -19.2% (P<0.01),respectively;The activities of exocellulase were increased by 3.4% (P>0.05),17.5% (P<0.01),-18.0% (P<0.01) and -25.7% (P<0.01),respectively;The activities of cellobiase were increased by 8.3%,23.7%,-24.9% and -31.8% (P<0.01),respectively;The activities of amylase were increased by 2.8%,8.6%,16.0% and 23.3% (P<0.01),respectively;The activities of pectinase were decreased by 11.8%,12.1%,11.2% and 11.1% (P<0.01),respectively;The activities of protease were decreased by 10.9%,30.4%,49.6% and 56.2% (P<0.01) respectively;However,the activities of xylanase did not show any significant changes (P>0.05).It was concluded that when the supplemental doses of paraformaldehyde was 300 mg/kg,the voluntary intake of sheep was the highest,and the protozoa number in rumen fluid was decreased,the total number of bacteria and the cellulase activity were the highest.

Energy Metabolism and Requirement of Jinjiang Cattle in the Latter Stage of Fattening
BAI Jun, ZHAO Erlong, LI Meifa, XIN Junping, XU Lanjiao, QU Mingren, YI Zhonghua, YANG Shitang, YANG Jianjun, LI Yanjiao
2019, 46(3):  732-739.  doi:10.16431/j.cnki.1671-7236.2019.03.011
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This experiment was conducted to investigate the energy metabolism and requirement of Jinjiang steers in the latter stage of fattening.The Jinjiang steers in the present study were chosen from our previous study,which were fed at different energy levels in the early stage of fattening (The NEmf of the five diets were 6.02,6.38,6.74,7.10 and 7.46 MJ/kg with 10 heads per group and test period was 116 d).After the early stage of fattening,the group were unchanged and the total of 35 remaining cattle with (355.94±35.11) kg average body weight in five group were re-continued for fattening feeding.The steers were fed 5 diets with different energy levels,which were formulated according to 100% (group A),106% (group B),112% (group C),118% (group D) and 124% (group E) of NEmf for the expected weight gain of 1.2 kg/d (China Feeding Standards of Beef Cattle (NY-T 815-2004)),respectively.The NEmf in five diets of groups A,B,C,D and E were 6.21,6.58,6.95,7.33 and 7.70 MJ/kg,respectively.The feeding test and digestion and metabolism test were carried out to measure the growth performance and energy metabolism indexes of Jinjiang steers,and then the models were built to predict the digestive energy requirement and metabolism energy requirement.The pre-test period was 10 d and test period was 128 d.The results showed as follows:①The total energy intake of Jinjiang steers at the latter stage of fattening of groups D and E was significantly reduced than that of other groups (P<0.05).②Group B had the highest energy utilization efficiency,with total energy digestibility and metabolic rate were 90.59% and 83.36%,respectively.③There was a positive linear correlation between average daily gain and digestive energy intake/metabolizable energy intake (R2=0.997,R2=0.993),the digestive energy requirement and metabolizable energy requirement of Jinjiang steers could be estimated by the following equations:DEm=0.770W0.75+40.088×ADG;MEm=0.645W0.75+38.603×ADG (DEm represented digestive energy requirement (MJ/d);MEm represented metabolizable energy requirement (MJ/d);W0.75represented metabolic body (kg);ADG represented average daily gain (kg/d)).The digestive energy requirement and metabolizable energy requirement for maintenance of Jinjiang steers were 0.770 and 0.645 MJ/(kg W0.75·d),respectively.Digestible energy and metabolizable energy required for per kilogram of body weight gain were 40.088 and 38.603 MJ,respectively.

pplication and Mechanism of Glucosamine in Regulating Birth Weight and Uniformity of PigletsA
FENG Tao, WEI Shangli, CAO Xin, TIAN Jianhui, LIU Yan
2019, 46(3):  740-746.  doi:10.16431/j.cnki.1671-7236.2019.03.012
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Litter size and birth weight are the two important factors for sows breeding productivity in pig industry,but litter size and birth weight show negative correlation.Recently,one of the research hot spots is how to improve birth weight and decrease its variation of piglets.Glucosamine is one of the feed stuffs and is widespread in nature.Published papers have showed that late pregnancy feed supplementation with glucosamine can regulate the birth weight and placenta development of piglets.This paper introduced the application effects of nutrients including amino acids and proteins,energy sources and functional additives on regulation of birth weight and variation of piglets,described the characteristics,source and synthetic method of glucosamine,mainly expounded the effect of glucosamine added in late pregnancy of sows on fructose concentrations stabilization in fetus,promotion placental development and placental function improvement,and analyzed the potential mechanisms of glucosamine regulation on placental development,birth weight and variation through increasing placental stroma development,improving placental folded bilayer extension,stimulating pig placenta trophectoderm cell proliferation,and enhancing placental function,and then raised the future research directions of glucosamine regulation on birth weight and variation in piglets.This review expected to supply a theoretical and practical basis for controlling birth weight and uniformity of piglets in pig industry.

Genetics and Breeding
Effect of lnc4351 Interference on the Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells
ZHANG Junxing, ZHU Feifei, LI Yan, CHEN Mingming, LIU Xinfeng, GUO Hong, DING Xiangbin
2019, 46(3):  747-755.  doi:10.16431/j.cnki.1671-7236.2019.03.013
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Based on the previous established isolation and culture of bovine skeletal muscle satellite cells and in vitro myogenic differentiation model,a high-throughput sequencing predicted lncRNA (lnc4351) with a great differentially expression during myogenic differentiation process was used as a target to explore the effect of lncRNA on the proliferation and differentiation of bovine skeletal muscle satellite cells,and the bioinformatics analysis and subcellular localization were performed in this study.The siRNA of lnc4351 was synthesized and transfected into bovine skeletal muscle satellite cells,the EdU staining was performed to detect the effect of interference with lnc4351 on cell proliferation.Simultaneously,an in vitro induced differentiation experiment was performed to observe the formation of myotubes after transfecting siRNA.To explore effects of interference with lnc4351 on cell differentiation,the mRNA and protein levels of the differentiation markers MyoG and MHC genes were detected using Real-time PCR and Western blotting,respectively.The results showed that lnc4351,located on chromosome 14 in cattle,was an unreported lncRNA with no protein coding potential.lnc4351 was distributed in the cytoplasm and nucleus of bovine muscle satellite cells,mainly in the nucleus.When the expression of lnc4351 was interfered,the ratio of EdU-positive cells was significantly decreased (P<0.05),suggesting that down-regulation of lnc4351 significantly inhibited the proliferation of bovine skeletal muscle satellite cells.In addition,the number of myotubes had an increasing trend and the protein levels of differentiation markers MyoG and MHC were significantly or extremely significantly up-regulated after interfering lnc4351 (P<0.05;P<0.01),indicating that down-regulation of lnc4351 could promote the myogenic differentiation process of bovine satellite cells.Down-regulating lnc4351 could inhibit the proliferation of bovine skeletal muscle satellite cells and promote its myogenic differentiation process.This study would provide a reference for further study of lncRNA on the regulation of bovine skeletal muscle development and related studies of muscle development.

Identification of Porcine circ0015292 and Its Expression Pattern in Ovarian Follicles
XIE Su, LI Mengxun, QIU Meiyu, GONG Hongbin, SUN Yishan, SUN Xiaomei, HUANG Tao
2019, 46(3):  756-763.  doi:10.16431/j.cnki.1671-7236.2019.03.014
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High throughput sequencing and bioinformatics were used to explore the role of circRNA in porcine follicular development and its possible mechanism,in order to provide the biological foundation for further exploring the regulatory mechanism of circRNA on porcine follicular development.Different levels of follicles and tissue samples from Meishan and Duroc pigs were collected,and then the total RNA was extracted.The differential expression of circRNA in follicles was screened by RNA-Seq technology.Meanwhile,the expression of RNA-Seq in the follicles of Meishan and Duroc pigs was validated and analyzed.At the same time,Real-time PCR was used to make tissue expression profiles.The results confirmed the existence of circ0015292,and it was expressed differently in muscle,heart,liver,spleen,lung,kidney,ovary,fallopian tube,uterus,pituitary gland and hypothalamus,and the expression level was relatively high in heart,spleen,lung and ovary.The expression level of circ0015292 in follicle S of Meishan pigs was extremely significantly higher than that of Duroc pigs (P<0.01),and the expression levels of follicles M1,M2 and L in Duroc pigs were significantly higher than those in Meishan pigs (P<0.05).The potential target genes of circ0015292 (GADD45G,CCR2,IL20RB,CFL1,LAMB3) were related to the development of porcine follicles.The results indicated that circ0015292 was expressed at different levels in different tissues and follicles at different stages;some target genes were involved in cell cycle and ovarian development-related signaling pathways,suggesting that circ0015292 might indirectly participate in follicular development through the regulation of target genes.This result would provide a new idea for the study of pig reproductive mechanism.

Condition of Adenovirus-mediated High Efficient Transgenic Embryo Production in Buffalo
LU Xingrong, PANG Chunying, DENG Tingxian, DUAN Anqin, MA Xiaoya, LIANG Shasha, LIANG Xianwei
2019, 46(3):  764-773.  doi:10.16431/j.cnki.1671-7236.2019.03.015
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In order to improve the production efficiency of transgenic buffalo embryos,this study was aimed to explore the optimal conditions of adenovirus-infected buffalo granular cells and embryos.Taking buffalo granule cells and embryos developed in vitro as research objects,buffalo granular cells were infected with 0,1×100,1×10-1,1×10-2,1×10-3,1×10-4,1×10-5 GFU/mL adenovirus to obtain the best infection concentration;The buffalo granular cells were infected with the optimum concentration condition for 24,48,72 and 96 h to obtain the optimal infection time,and the results were observed by inverted fluorescence microscope.The optimal concentration and time of adenovirus-infected to buffalo granular cells were infected to the embryos in 2- and 4-cells stages,respectively,the optimal conditions of embryo infection were explored,and the embryo cleavage rate,blastocyst rate and transfection blastocyst rate were analyzed.The results showed that the optimal adenovirus infection efficiency of buffalo granular cells could be obtained by 1×10-2 GFU/mL concentration and 48 h infection time of adenovirus.The embryos in 2- and 4-cells stages without zona pellucida and non-complete zona pellucida were infected with adenovirus got green light,while there was no light in the complete zona pellucida embryo.The embryos in 2-cell stage stopped developing after infection,while the incomplete zona pellucida embryos in 4-cell stage could continue developing into blastocyst.The blastocyst rate and blastocyst transfection efficiency of incomplete zona pellucida group was superior to other groups after infected with adenovirus in 4-cell stage.In summary, the incomplete zona pellucida,1×10-2 GFU/mL,48 h,and 4-cell stage infection contributed to efficient expression of the target gene in buffalo embryo,and then improved the efficiency of transgenic buffalo embryo production.

Polymorphism of PRLR and FSHβ Genes and Its Association Analysis with Reproductive Traits in Pigs
FAN Yiping, WANG Xiaomei, JIA Qitao, LI Chengbo, LIANG Xiaojuan, CAO Chunwei, ZHOU Lei, TAO Cong, ZHAO Jianguo, LIU Jianfeng, WANG Yanfang
2019, 46(3):  774-781.  doi:10.16431/j.cnki.1671-7236.2019.03.016
Abstract ( 201 )   PDF (1818KB) ( 148 )  
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This study was aimed to investigate the polymorphism of prolactin receptor (PRLR) and follicle-stimulating hormone β (FSHβ) genes and its association with the reproductive traits in sows.The polymorphism of PRLR and FSHβ genes in 487 sows of Large White,Duroc and Landrace pigs was detected by PCR-RFLP method,and the association between different genotypes and reproductive traits such as total litter size,litter size,weaning litter weight and weaning litter size were analyzed by One-Way ANOVA analysis and LSD method.The results showed that B allele of PRLR gene was predominant allele in Large White and Landrace pigs,and the gene frequency were 0.717 and 0.548,respectively.The total number born of BB genotype in Large White pig was significantly higher than that of AA genotype (P<0.05),the weaning litter weight of BB genotype in Landrace pig was significantly higher than that of AB genotype (P<0.05).B allele of FSHβ gene had a high frequency in Large White,Duroc and Landrace pigs,and the gene frequency were 0.804,0.760 and 0.789,respectively.The total number born and number born alive of BB genotype in Large White pig was significantly higher than that of AB genotype (P<0.05),displaying BB>AA>AB trend.In conclusion,the polymorphism of PRLR and FSHβ genes significantly affected the reproductive traits of sows in Rongchang pigs farm,and they could be considered as candidate selective markers for molecular breeding of sows.

Cloning and Sequence Analysis of Porcine SMYD3 Gene and Its Effects on Proliferation of Porcine Fibroblasts
SHE Chun, ZHANG Yan, ZHANG Caiyu, YIN Guijun, SHI Deshun, LI Xiangping
2019, 46(3):  782-791.  doi:10.16431/j.cnki.1671-7236.2019.03.017
Abstract ( 163 )   PDF (2523KB) ( 141 )  
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This experiment was aimed to clone and analyze the sequence of porcine SET and MYND domain-containing protein 3 (SMYD3) gene,and study its effect on the proliferation of porcine fibroblasts.Firstly,SMYD3 gene was cloned,according to the siRNA and shRNA sequences of other species SMYD3 gene,two shRNA sequences of SMYD3 gene were obtained by homologous alignment analysis,pSicoR-GFP-SMYD3 shRNA1/shRNA2 expression vector were constructed and transfected into HEK293T cells.The inhibition efficiency of shRNA was analyzed by Real-time PCR,and screened out shRNA with better inhibition efficiency.Then the pLVX-IRES-ZsGreen1-SMYD3 and pSicoR-GFP-SMYD3 shRNA eukaryotic expression vectors were constructed,the proliferation of porcine fibroblasts was analyzed,and the expression levels of Nanog,DNMT1 and DNMT3a genes were detected by Real-time quantitative PCR.The results showed that the cloned CDS length of porcine SMYD3 gene was 1 404 bp.Bioinformatics analysis results showed that the homology of the SMYD3 gene was 99.5% between pig and Sus scrofa,which shared 93.8% and 92.9% identity with Capra hircus and Bos mutus,respectively.The expression of SMYD3 gene was significantly inhibited by shRNA1/shRNA2 (P<0.05),and the inhibitory rates were 34% and 54%,respectively.pSicoR-GFP-SMYD3 shRNA2 was selected for follow-up studies.The pLVX-IRES-ZsGreen1-SMYD3 and pSicoR-GFP-SMYD3 shRNA eukaryotic expression vectors were constructed,and clear green fluorescent signal was observed when the plasmids were transfected into HEK293T cells by liposome method.The results of lentiviral infection and Real-time quantitative PCR showed that compared with the negative and blank control groups,overexpression of SMYD3 gene promoted the porcine fibroblast proliferation,and the expression of Nanog and DNMT1 genes were significantly increased (P<0.05);And inhibition of SMYD3 gene inhibited porcine fibroblasts proliferation while the expression of Nanog,DNMT1 and DNMT3a genes were significantly decreased (P<0.05).This results indicated that the expression of SMYD3 gene was significantly associated with the proliferation of porcine fibroblasts.

Association Between Polymorphism of MC4R and CDC16 Genes and Growth Traits in Large White Pigs
ZHAO Congzhe, LUO Xiaotong, LI Zhaohua, ZHANG Zhibin, YU Yongsheng
2019, 46(3):  792-799.  doi:10.16431/j.cnki.1671-7236.2019.03.018
Abstract ( 180 )   PDF (1244KB) ( 104 )  
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This study was aimed to detect the polymorphism of melanocortin-4 receptor (MC4R) and cell division cycle protein 16 (CDC16) in Large White pigs by HRM typing,and analyze its association with growth traits in Large White pigs.A total of 246 Large White pigs ear tissues were collected as research objects,DNA was extracted using the AxyPrep Genomic DNA Extraction Kit.After PCR amplification of small population samples,the polymorphic positions of MC4R and CDC16 genes in the population were searched by sequencing.According to the findings,the polymorphic sites were designed for HRM-specific primers.The HRM method was used to detect the polymorphism in large populations,and the results were correlated with growth traits in Large White pigs.The results showed that one mutation site 2207A>G,two alleles (A and G) and three genotypes (AA,AG and GG) were detected in the MC4R gene sequence.There was a mutation site 837T>C,two alleles (T and C),two genotypes (TT and CT) in the 18th exon of CDC16 gene.The χ2 test results showed that the genotype frequency and gene frequency of MC4R and CDC16 genes were not significant difference between the two populations,which was consistent with the Hardy-Weinberg equilibrium (P>0.05).The polymorphic information content (PIC) result showed that MC4R gene was moderate polymorphic (0.25<PIC<0.5),and CDC16 gene was low polymorphic (PIC<0.25) in both populations.The correlation analysis results showed that MC4R gene had significant effects on body weight and daily gain in Large White pigs (P<0.05),CDC16 gene had significant effects on body height,body length and body weight (P<0.05).In summary,MC4R and CDC16 genes had certain effects on the growth and development of Large White pigs,which provided a reference for the modern molecular breeding of meat quality and growth traits in Large White pigs.

Effect of RNA Interference Buffalo MTPN Gene on Proliferation, Apoptosis of Granulosa cells and Hormone Secretion
CHEN Chao, LI Jun, HUA Liping, AN Zhigao, HU Xiangwei, LIU Shenhe, YANG Liguo
2019, 46(3):  800-807.  doi:10.16431/j.cnki.1671-7236.2019.03.019
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This study was aimed to investigate the effect of MTPN gene knockdown on proliferation and apoptosis of buffalo granulosa cells in vitro and estrogen and progesterone secretion.The expression of MTPN gene in granulosa cells was knocked down by RNAi technology.The expression of MTPN gene and proliferation and cell cycle-related genes were detected by Real-time PCR,the cell proliferation and cell cycle distribution were detected by CCK-8 assay and flow cytometry,respectively.The estrogen and progesterone levels in the cell culture medium were determined by ELISA kit.The results showed that the relative expression of MTPN gene was decreased by 60% after siRNA (si-MTPN) transfection (P<0.01).The cell proliferation was significantly inhibited (P<0.05),the number of cells in G1 phase decreased,and the number of cells in S phase increased,the number of cells in G2 phase extremely significantly increased (P<0.01),and the cells were blocked in G2 phase.The expressions of Cyclin D2 and Cytochrom C genes were significantly up regulated (P<0.05),and the expressions of Caspase9 and Fas genes were extremely significantly up regulated (P<0.01).ELISA results showed that the levels of estrogen and progesterone secretion were significantly decreased (P<0.05).In conclusion,knockdown of MTPN gene could inhibit the proliferation of buffalo granulosa cells in vitro and the secretion of estrogen and progesterone by regulating the expression of related genes,which laid a reference for elucidating the molecular mechanism of MTPN gene involved in follicular development of livestock.

Research Progress on the Genetic Polymorphisms of DGAT1 Gene and Its Association with Economic Traits in Chinese Cattle Breeds
WANG Siwei, WANG Xueqing, WANG Kun
2019, 46(3):  808-817.  doi:10.16431/j.cnki.1671-7236.2019.03.020
Abstract ( 224 )   PDF (1062KB) ( 122 )  
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DGAT1 is the only key enzyme in the synthesis of triglyceride,which plays an important role in animal fat metabolism and lipid deposition in tissues.Therefore,DGAT1 gene,which coded DGAT1,has become to one of the most important candidate genes for studing lactating traits in dairy cows and economic traits in beef cattle.In recent years,with the deepening of relevant research,researchers generally believe that DGAT1 gene has a significant effect on milk yields,milk fat percentage,milk protein rate,lactose rate,milk fatty acid and fertility traits such as average barren days,number of sperm and the age at first calving.The correlation between DGAT1 gene and body fat of beef cattle has also been proved.Therefore,the polymorphisms of DGAT1 gene in different cattle breeds and populations and its association with economic traits has been studied.This paper mainly reviews the genetic polymorphisms of DGAT1 gene and its association with economic traits of dairy cows,beef cattle,buffalo and yak in various regions of China.It has been proved that DGAT1 gene has an important regulatory effect on the lactating traits of dairy cows and the meat quality and growth traits of beef cattle.However,the effects of DGAT1 gene genetic polymorphism was different in different regions and populations due to environmental conditions,breeds and breeding background.Therefore,in order to apply the DGAT1 gene to the breeding,it is necessary to clarify the specific genetic effects of the gene on the research population and make a comprehensive analysis with other candidate genes.

Study on the Change Law of Reactive Oxygen Level and the Expression of Various Antioxidant Enzymes mRNA in Different Developmental Stages of Porcine Corpus Luteum
GAO Jianhong, LI Chuang, QIAO Jiaqi, FANG Nanzhu, LI Zhongshu, LIU Haixing
2019, 46(3):  818-823.  doi:10.16431/j.cnki.1671-7236.2019.03.021
Abstract ( 186 )   PDF (1754KB) ( 233 )  
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The purpose of the experiment was to study the change law of reactive oxygen species (ROS) and the expression of antioxidant enzymes genes in the development and degradation of porcine corpus luteum,and to supplement the theoretical basis and provide new ideas for interpreting the antioxidant mechanism of porcine corpus luteum.The ovaries used in the experiment were collected from Yanji slaughterhouse.After removed from the ovary,the corpus luteum was initially divided into early corpus luteum,middle corpus luteum,late corpus luteum and white bodies by its size.The middle and late corpus luteum were then accurately separated by detecting progesterone levels.The levels of ROS in the corpus luteum were detected by frozen section and DHE fluorescence staining technique.The amount of expression of manganese superoxide dismutase (Mn-SOD),catalase (CAT) and Glutathione peroxidase (GPx) of antioxidant enzymes were examined by Real-time PCR in each period.The results showed that the ROS level of porcine corpus luteum increased regularly with the periodic development of porcine corpus luteum,that was,ROS level in the late period were significantly higher than that in other periods (P<0.05).And the ROS level in the middle period was significantly higher than that in the initial period and white body (P<0.05),and there was no significant difference between white body and the initial period (P>0.05).The expression of Mn-SOD, GPx1 and GPx4 decreased regularly,they were significantly higher in the initial period and white body than that in the middle and late stage (P<0.05),and their expression in the middle stage was significantly higher than that in the late stage (P<0.05).and the white body was significantly higher than that in other stages (P<0.05).The expression of GPx3 gene in white body was significantly higher than that in other stages (P<0.05),the expression in initial period was significantly higher than that in the middle and later stage (P<0.05),and the expression in the middle stage was significantly higher than that in the late stage (P<0.05).The CAT expression had no significant change with the development of the corpus luteum (P>0.05).In conclusion,ROS,Mn-SOD and GPx were the influencing factors for the development and degradation of corpus luteum,and the level of ROS was opposite to that of Mn-SOD and GPx.

Comparative Transcriptome Analysis of Lactation Pituitary Gland in Yak
ZHU Yuxing, LIU Jun, AI Yi, CHEN Tong, DENG Zhihe, HE Shiming, WU Jinbo, LAN Daoliang
2019, 46(3):  824-831.  doi:10.16431/j.cnki.1671-7236.2019.03.022
Abstract ( 185 )   PDF (903KB) ( 103 )  
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This study was aimed to explore the transcriptome difference of pituitary gland in yak with different milk production during lactation,and provide a reference for further elucidating the mechanism of lactation pituitary gland in yak.The total RNA was extracted from pituitary gland tissue,high-throughput sequencing and analysis of pituitary tissue transcriptome during high lactation calf and low-yield calf lactation were performed using RNA-Seq technology.By the comparison and analysis of the transcriptome data for the pituitary gland tissue of high-yielding yak and low-yielding yak,114 differential genes were screened out,of which 55 were up-regulated and 59 were down-regulated.Further functional analysis indicated that these differential expressed genes involved in multiple classes based on GO analysis and KEGG pathways.The GO analysis result showed that the functions of the differential genes were closely related to the amino acids metabolism and other biological processes.KEGG pathway analysis result revealed that these genes were mostly enriched in the pathway of cell adhesion molecule pathway.Moreover,several other pathways related to immune such as Staphylococcus aureus infection and leishmaniasis also showed significant enrichment.The pituitary gland transcriptome of high-yielding yak and low-yielding yak during lactation had been compared,and the related differentially expressed genes had been screened and analyzed,which provided new ideas for improving the milk production of yaks.

Preventive Veterinary Medicine
Eukaryotic Expression of Recombinant Baculovirus of PEDV Isolated Strain S1 Gene
HE Haijian, WU Yuan, WANG Luyan, LI Qunjin, BA Shaobo, SHI Lin, SHAO Chunyan, SUN Jing, JIANG Sheng, WANG Xiaodu
2019, 46(3):  832-839.  doi:10.16431/j.cnki.1671-7236.2019.03.023
Abstract ( 207 )   PDF (2558KB) ( 202 )  
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In order to study the eukaryotic expression and reactivity of porcine epidemic diarrhea virus (PEDV) isolated strain (PEDV/LA/2014/02) S1 gene,the recombinant protein (His-PEDV-S1) was expressed by the baculovirus expression system.The rare codon of PEDV S1 gene in sf9 cells was analyzed using online software,and the optimized gene was synthetized by biotechnology company.PEDV S1 gene were cloned into the baculovirus shuttle vector (pFastBac HT A),the PEDV S1 gene was recombined into baculovirus genome in Esherichia coli DH10Bac cells and identied by PCR.The PEDV-S1 recombinant successfully baculovirus genome was transfected into sf9 cells.The packaging viruses were inoculated into sf9 cells,the cytopathic effect was observed by optical microscope.The mRNA level of PEDV S1 gene in sf9 cells was analyzed by RT-PCR.The expression and antigenicity of recombinant PEDV S1 were verificated by SDS-PAGE and Western blotting.The results showed that the pFastBac HT A-PEDV-S1 (pSL598) was successfully constructed,the recombinant baculovirus expressing His-PEDV-S1 was packaged,the sf9 cells became big and round,the vacuoles appeared in cytoplasm after baculovirus infection.The mRNA of PEDV S1 gene was expressed in baculovirus infected cells.The recombinant protein His-PEDV-S1 was mainly expressed in sf9 cells precipitation,and the size of the protein was about 83 ku.The His-PEDV-S1 had good reactivity of mouse anti-His antibody and swine anti-PEDV positive antisera.This study provided materials for the development of a new subunit vaccine for the new PEDV strain and the prevention and control of PEDV strain.

Establishment and Evaluation of the Artificial Infection Model of Streptococcus suis Type 2 in Kunming Mice
LI Xiuli, DONG Zhimin, YAN Xiaocui, TIAN Xiangxue, YANG Chunlei, CHI Jingjing, REN Weike, LI Fuqiang, TAN Shixu, YAN Minghua
2019, 46(3):  840-848.  doi:10.16431/j.cnki.1671-7236.2019.03.024
Abstract ( 194 )   PDF (4273KB) ( 190 )  
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In order to establish a stable model of Streptococcus suis type 2 (SS2) artificial disease model,the Kunming mice were infected with the SS2 Y15293 strain which was isolated from a swine farm in Tianjin by intraperitoneal injection,and the model were evaluated by clinical symptoms observation,histopathological detection and bacterial isolation and identification test.The results showed that the LD50 of the SS2 Y15293 strain was 6.7×107 CFU/mL for the Kunming mice.The Kunming mice were attacked with one dose of LD50 of SS2 Y15293 strain,and the mice of the test group showed the spirit was depressed,the hair was inverted,and the eyes had secretions in 24 h post infection,and then showed head and neck tilt,tumbling,tremor and other neurological symptoms at 72 to 96 h post infection.The peak of death appeared at 48 to 72 h post infection.In the three repeated tests,the morbidity and mortality of mice in the experimental group were 100%,50% to 70%,and the occurrence rate of neurological symptoms was 20% to 30%.Compared with control group,the intake and body weight of the mice in test group were significantly decreased (P<0.05),and the score of clinical symptoms obviously increased (P<0.05).Histopathological examination showed that heart and lung tissues of mice in test group had different degrees of hemorrhage and inflammatory cell infiltration,and meningitis,intraventricular hemorrhage,and brain tissue were filled with inflammatory cells.Bacterial regression test results showed the bacterial colonization was found in brain,heart,liver,spleen,lung and kidney of dead mice in test group,and the morphological,staining and molecular biological characteristics of the isolates from mice of test group were consistent with the SS2 Y15293 strain.It was confirmed that the SS2 Y15293 strain could infect Kunming mice and cause the clinical symptoms.The results of this study suggested that the SS2 Y15293 strain infected Kunming mice could be used as a candidate artificial infection model for SS2.

Preparation of Monoclonal Antibodies Against Phenylethanolamine A and Establishment of ELISA Method
XIANG Zilai, WANG Ling, XIA Sugan, ZOU Hui, GU Jianhong, YUAN Yan, LIU Xuezhong, LIU Zongping, BIAN Jianchun
2019, 46(3):  849-857.  doi:10.16431/j.cnki.1671-7236.2019.03.025
Abstract ( 158 )   PDF (1737KB) ( 262 )  
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To prepare monoclonal antibodies against phenylethanolamine A (PA) and establish a fast,simple and sensitive method for the detection of PA,a derivative of PA was coupled with bovine serum albumin (BSA) and ovalbumin (OVA) as immunogen and coating antigen by diazotization.The immunogen that emulsified by Freund’s adjuvant was used for immunization of 6 to 8 weeks’ BALB/c mice in accordance with conventional procedures.The immune spleen cells of mice with high serum antibody titer were fused with SP2/0 myeloma cells by PEG method.After three subclonal screening,a hybridoma cell strain D6H8 of secreting anti-PA monoclonal antibody was screened out and the inducing ascites in vivo method was employed to produce monoclonal antibodies.The McAb was identified to be IgG1 subtype and kappa light chain.The purified antibody showed no cross-reactions with salbutamol amine,clenbuterol hydrochloride,isoprenaline hydrochloride and deoxyepinephrine hydrochloride (CR<0.18%),indicating that the antibody had good specificity.An indirect competitive ELISA method for PA drug residues was established using this antibody.The results showed that the titer of ascites antibody was 1∶12 800 and the linear relationship was good when the concentration of PA in pork was 5 to 1 000 ng/mL.The linear equation was y=0.3861x-0.1845 (R2=0.990).The concentration of IC50and LOD were 58.88 and 3.83 ng/mL,respectively.The recovery rates were between 85.96% and 104.32%,indicating that the ELISA method was reproducible and stable.In summary,an indirect competitive ELISA method for the determination of PA residues had been successfully established with high sensitivity and good stability.

Optimization About Proliferated Condition of Newcastle Disease Virus LaSota Strain in BHK-21 Cells
DONG Bingmei, SUN Peijiao, MIAO Lizhong, SHEN Zhiqiang, HAN Wenyu
2019, 46(3):  858-864.  doi:10.16431/j.cnki.1671-7236.2019.03.026
Abstract ( 271 )   PDF (3768KB) ( 213 )  
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The aim of this study was to optimize the proliferation conditions of Newcastle disease virus (NDV) LaSota strain on BHK-21 cells,and provide reference for the application of cell line to produce NDV.Firstly,the tolerant concentration of BHK-21 cells to TPCK-trypsin was determined.On this basis,different concentrations of TPCK-pancreatin and newborn bovine serum were added into the medium respectively,and the pH of medium was regulated.The NDV LaSota strain was cultured in the medium and the optimal trypsin concentration,serum concentration and pH of the medium were determined respectively through measuring half-infection amount (TCID50) of the culture.On the other hand,the same quantity of NDV LaSota strain were inoculated to different number of cells or the same number of cells were inoculated by different quantity of virus.The optimal number of BHK-21 cell and the optimal dose of NDV LaSota strain were determined by measuring TCID50.Secondly,the proliferation curve was drawn to determine the optimal time for collection after inoculating the NDV LaSota strain in BHK-21 cells and the TCID50 of the supernatant was determinated at different time.Finally,the NDV LaSota strain was cultured in spinner flask and the TCID50,the half egg infection amount (EID50) and the serum agglutination price (HA) were measured.The results showed that the optimal TPCK-trypsin concentration was 3 μg/mL,the optimal toxic dose was 106.375 EID50,the number of cells was 1×106,the pH of the medium was 7.2,and cultured in serum-free medium when the NDV LaSota strain was inoculated in BHK-21 cells.The optimum harvested time was from 34 to 36 h after BHK-21 cells were inoculated with NDV LaSota strain.At the end,the proliferation of NDV LaSota strain was carried out using spinner flask.The results showed that the cell culture solution had a TCID50 value of 107.0/0.1 mL,the EID50 was 107.5/0.1 mL,and the HA titer was 1∶256.Therefore,the application of the above conditions to culture NDV LaSota strain in BHK-21 cells was fully applicable to the large-scale production of the NDV,and in line with the current veterinary biological products and inspection manufacturing regulations.

Effect of L-NMMA on Weight Gain of Chickens Infected with Chicken Coccidia Premature Attenuated Vaccine
FENG Yuping, HAN Tianfei, LYU Qianghua, MI Chenglong, HAO Feifei, ZHANG Li, ZHANG Xuesong, ZHENG Mingxue
2019, 46(3):  865-872.  doi:10.16431/j.cnki.1671-7236.2019.03.027
Abstract ( 189 )   PDF (2823KB) ( 135 )  
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In order to study the effect of chicken coccidia premature attenuated vaccine and nitric oxide synthase inhibitor (L-NMMA) on the weight gain of immunized chicken,and the effect of L-NMMA on immune efficacy.Forty SPF chicks were divided into two blank control groups (C1 and C2) and two single-dose repeated inoculated groups(T1 and T2).In groups T1 and T2,chicks were inoculated repeatedly with the premature attenuated vaccine of chicken coccidia at the recommended single dose,then the chicks with empty stomach were weighted separately on 7th and 14th after the second inoculated.The duodenum,jejunum and cecum lesions were recorded.The heart,liver,spleen,lung,kidney,glandular stomach,muscular stomach,duodenum,jejunum and cecum of the chicks were collected for histopathological examination.Another forty SPF chicks were divided into blank control group (C20),challenge control group (T21),immune control group (T22) and immune with drug group (T23).In groups T22 and T23,chicks were vaccinated twice with premature attenuated vaccine at the recommended immune dose,and the chicks in T23 group were injected with L-NMMA at the same time.On the 14th after the second immune,the chicks were gavaged with mixed virulent chicken coccidia,then the average weight gain,relative weight gain,intestonal (duodenun,jejunum and cecum) lesion scores,reduction of lesion scores (RLS),bloody stool scores,mortality and oocyst production were measured.The results showed that the average weight gain of the vaccinated group was significantly lower than the unvaccinated group (P<0.05).The duodenum,jejunum and cecum of the vaccinated group had mild lesions and the mucosal epithelial cells had different degrees of damage and shedding.The average weight gains of the vaccinated with L-NMMA group after the first,second immuned and challenged were all extremely significantly higher than the immune-challenged control group (P<0.01),while the average weight gain was not significantly different with the blank control group (P>0.05),and there were no significant differences in intestinal lesion score and oocyst yield between immune control group and drug immune group (P>0.05).The results demonstrated that premature attenuated vaccine of chicken coccidia had light damage to the intestines of the chicks and had certain negative effects on the weight gain.In addition,L-NMMA could reduce the vaccine’s adverse effect on the weight gain and had no effect on the immune efficacy.

Establishment and Application of Duplex PCR for Rapid Identification of Staphylococcus and Streptococcus of Livestock and Poultry in Guizhou Province
ZHANG Yundan, MA Guangqiang, YUE Jun, ZHOU Bijun, WEN Ming, CHENG Zhentao
2019, 46(3):  873-880.  doi:10.16431/j.cnki.1671-7236.2019.03.028
Abstract ( 214 )   PDF (2246KB) ( 102 )  
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In order to establish a duplex PCR method for accurately and quickly identifying common pathogens Staphylococcus and Streptococcus in clinical cases of livestock and poultry,this experiment selected nuc gene of Staphylococcus and EF-TU gene conserved fragments of Streptococcus to design a pair of specific primers,construct a duplex PCR system which could simultaneously identify Staphylococcus and Streptococcus,optimize the reaction conditions,screen out the optimal primer concentration and annealing temperature;Using this method to detect 73 other gram-positive clinical isolates,the specificity of the method was evaluated;The sensitivity of the detection method was determined by diluting and counting the cultured Staphylococcus and Streptococcus;The detection method was used to detect the clinical isolates of Staphylococcus and Streptococcus of livestock and poultry in Guizhou province.The results showed that the optimal primer addition amount was 1 μL,and the optimal annealing temperature was 56 ℃.The other 73 strains showed no amplification bands,and the duplex PCR method was better.The specificity of the sensitivity test showed that the sensitivity of Staphylococcus and Streptococcus were 1.50 ng/μL and 1.44 pg/μL,respectively.The retest results of clinical samples showed that there were 40 strains of Staphylococcus (54.79%) and 33 strains of Streptococcus (45.21%) in 73 clinical isolates,the coincidence rate with traditional bacteria separation and identification method was 97.26%.This experiment established a duplex PCR method for specific,sensitive and rapid identification of Staphylococcus and Streptococcus pathogens in Guizhou province,providing effective techniques for rapid diagnosis and epidemiological investigation of clinical cases.

Isolation and Identification of Two Pseudorabies Virus Variants and Analysis of Molecular Characteristics of gB,gC and gE Genes
LI Xiang, GUO Zhenhua, RUAN Haiyu, QIAO Songlin, ZHANG Yifang, ZHANG Gaiping
2019, 46(3):  881-890.  doi:10.16431/j.cnki.1671-7236.2019.03.029
Abstract ( 197 )   PDF (5920KB) ( 104 )  
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To investigate the genetic variation of pseudorabies virus (PRV) in Henan province in recent years,in this study,the virus isolation and identification were performed by cell blind transmission,indirect immunofluorescence,Western blotting,plaque purification and transmission electron microscopy in brain tissue samples from suspected pseudorabies farms in Luohe and Zhongmou in 2017.The viruses titer and growth curve of the isolated strains were determined by 50% tissue culture infective dose (TCID50),and the lethality of the mice was determined by mouse infection test.Futhermore,the gB,gC and gE genes were aligned and analyzed.Two strains of PRV were successfully isolated and identified by indirect immunofluorescence,plaque purification and transmission electron microscopy,named as HeN-LH strain and HeN-YM strain,respectively.The titers of HeN-LH and HeN-YM strains reached 108.35 and 106.63 TCID50/mL at 36 h post infection on PK-15 cells.The lethality of the mice showed that the median lethal dose (LD50) of HeN-LH and HeN-YM strains was 102.13 and 103.25 TCID50,respectively.The evolutionary analysis displayed that the two isolates were closer to the PRV variants which were mainly epidemic in China since 2011.Amino acids sequences alignment showed that,similar to other variants,gB,gC and gE genes had multiple amino acid variations,and there were unique amino acid insertions and deletions at specific sites.In short,we successfully isolated and identified two PRV variants,and confirmed the current pseudorabies were mainly caused by the PRV variants,and the results could provide data for the prevention and control of pseudorabies and the selection of vaccine strains in Henan province.

Isolation,Identification and Biological Characterization Analysis of One Strain Canine Parainfluenza Virus
LIU Chang, QIN Tong, LIANG Lin, YOU Xinyue, WANG Chunfeng, QIAN Aidong, LI Jinxiang, CUI Shangjin
2019, 46(3):  891-897.  doi:10.16431/j.cnki.1671-7236.2019.03.030
Abstract ( 210 )   PDF (2981KB) ( 95 )  
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The aim of this study was to isolate and identify the canine parainfluenza virus (CPIV),and analyze its biological characteristics.CPIV positive canine lung tissue was inoculated into Vero cells.CPE were observed after four blind passages,then the cell culture fluid after 72 h was assayed for further RT-PCR identification,electron microscopy,HA and biological characteristic experiments.At the same time,N gene of CPIV was amplified,and phylogenetic tree was constructed.The result showed that one CPIV was successfully isolated and named as CPIV-BJ01.The result of RT-PCR showed that there was a specific target fragment of 534 bp.The electron microscopic observation showed that the virus were circular,enveloped and between 80 to 200 nm in diameter.It could agglutinate 1% porcine erythrocytes at 4 or 37 ℃.The virus was sensitive to high temperature and UV irradiation.The one-step growth curve showed that the virus replicated at a high speed between 12 to 48 h.Compared with 19 representative CPIV N genes,the nucleotide sequence homology analysis was 97.1% to 99.8%.The CPIV-BJ01 strain was closely genetically related to strain PIV5 1168-1 (GenBank accession No.:KC237064.1) and PIV5 ZJQ 221(GenBank accession No.:KX100034.1).

Study on the Immune Efficacy of A Trivalent Inactivated Vaccine Against Newcastle Disease, Infectious Bronchitis and Infectious Bursal Disease in Different Days Old Chicks
SHEN Jia, SHI Aihua, ZHANG Zhenhua, LI Lin, JING Xiaodong, ZHANG Jianwei, ZHENG Xiaolan, JIANG Beiyu
2019, 46(3):  898-904.  doi:10.16431/j.cnki.1671-7236.2019.03.031
Abstract ( 206 )   PDF (704KB) ( 279 )  
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To evaluate immune efficacy and immune duration of a trivalent inactivated vaccine against Newcastle disease (ND),infectious bronchitis (IB) and infectious bursal disease (IBD),the vaccine was administered to SPF and common healthy chicks with maternal antibodies at age of 7,14 and 21 days old.The blood samples of the chicks were collected at 21 d after vaccination,then ND haemagglutination inhibition antibody (HI Ab),IB HI Ab and IBD neutralizing antibody (NA) of the blood samples were examined,and virulent IBDV challenge were carried out.The results showed that the ND HI Ab,IB HI Ab and IBD NA titers of SPF chicks at 7 days old were 7.9log2,6.9log2 and 14.1log2,respectively,and the older the SPF chicks,the higher the antibody level.Three months after immunization,the ND HI Ab,IB HI Ab and IBD NA titers of chicks immused with 3 mL vaccine reached 6.5log2,6.1log2 and 13.6log2 respectively,and the protection rate against IBDV challenge was 100% (10/10).The immune effect of common chicks was almost similar with SPF chicks,the antibody levels increased with the age of the chicks,and the protection rate against IBDV was 100% (10/10).The results confirmed that the different days old chicks inoculated with the trivalent inactivated vaccine could produce good immunity to against virulent IBDV challenge,the immunity duration of the triple inactivated vaccine was 3 months at least.

Effect of Non-antigen Protein of Foot-and-Mouth Disease Vaccine on Immune Effect of 146S Antigen
LI Ming, ZHANG Yana, GUO Xiaoyu, GAO Xintao, LI Jinxiang, ZHU Hongfei
2019, 46(3):  905-912.  doi:10.16431/j.cnki.1671-7236.2019.03.032
Abstract ( 187 )   PDF (857KB) ( 100 )  
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In order to explore the effect of non-antigen protein on the immune effect of foot-and-mouth disease virus type A (FMDV-A) 146S antigen,mice and pigs were used as experimental animals.5 groups of mice for injection were prepared:Group A (3 μg 146S antigen),group B (3 μg 146S antigen+25 μg non-antigenic protein),group C (3 μg 146S antigen+50 μg non-antigenic protein),group D (3 μg 146S antigen+100 μg non-antigenic protein) and group E (blank control,PBS).At the same time,four groups of pig injection samples were prepared:Group A (18 μg 146S antigen),group B (18 μg 146S antigen+400 μg non-antigenic protein),group C (18 μg 146S antigen+4 000 μg non-antigenic protein) and group D (blank control,PBS).The cell immune response levels of mice in different vaccine sample groups were compared by lymphocyte proliferation assay and Real-time PCR.The level of humoral immune response produced by the animals (mice and pigs) of each test group was detected and evaluated by a liquid phase blocking ELISA method.The results of mice test showed that the average antibody level of group C was the highest in four test groups at the three test time points after immunization,and the average antibody titers were 4.13,5.83 and 5.50,respectively,while the antibody of group D was the lowest,the average antibody titer average antibody titers were 3.46,5.16 and 4.46 in the antibody detection,respectively;The cell proliferation ability and Th-1 type cytokine IL-6,IFN-β and TNF-α mRNA expression in group C were higher than other groups.The results of pigs test showed that the average antibody level between groups A and B was not significantly different (P>0.05),but the average antibody titers of groups A and B were extremely significantly higher than that of group C (P<0.01).The above results indicated that a small amount of non-antigenic protein had no effect on the immune effect of FMDV-A 146S antigen within a certain range,while a high concentration of non-antigenic protein inhibited the immune effect of FMDV-A 146S antigen.

Basic Veterinary Medicine
Analysis of Virulence Factors and Multidrug Resistance of Enterotoxin-producing Escherichia coli in Guangxi
GE Chenling, SHI Dali, HU Wen, SONG Xingxing, GE Qiang, MO Yupeng, HU Chuanhuo, WANG Xiaoye
2019, 46(3):  913-923.  doi:10.16431/j.cnki.1671-7236.2019.03.033
Abstract ( 217 )   PDF (2643KB) ( 112 )  
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This study was aimed to investigate the possible pathogens and the main causes of disease in a pig farm in Guangxi.The bacteria were isolated and identified by bacterial separation and purification,morphological identification,growth curve measurement,biochemical analysis and drug susceptibility test.The strain 16S rRNA,virulence genes and drug resistance genes were detected by PCR.The results showed that the strain isolated from the disease material was a Gram-negative bacterium with no spores,pili,and round and short rods at both ends,which had strong reproductive ability and rapid growth,and was suspected to be Escherichia coli (E.coli).The 16S rRNA amplification results showed that a 1 474 bp band appeared,and the sequencing results showed that the homology of the isolate with E.coli was 99%.The strain showed strong resistance to 31 antibacterial drugs gentamicin,tilmicosin,tetracycline,enrofloxacin,penicillin,lincomycin,sulfamethoxazole,etc.,all showed higher levels of drug resistance,and the drug resistance rate was as high as 87.10% (27/32).It was only sensitive to two drugs,amikacin and colistin.Toxicity factor detection identified a total of five virulence factors,FyuA,enterotoxin STb and LT,adhesin F41 and K88.The detection rate was 35.70% (5/14).It had been amplified β-lactamases blaTEM and blaOXA,tetracycline tet(A),sulfonamide Sul2,aminoglycosides aadA1 and aac(3')-Ⅱa,quinolone GyrA,GyrB and ParC,amide alcohol floR 10 drug resistance genes,the detection rate was 66.70% (10/15),and the detection of drug resistance genes was positively correlated with drug resistance phenotype.In summary,the strain presented a variety of complex virulence factors,complex drug-resistance,and serious multi-drug resistance,and corresponding precautions need to be taken to prevent spread and spread.

The Free Radical of Nitric Oxide Inhibits Porcine Circovirus Type 2 Replicion in vitro
LI Jizong, ZHANG Dongxia, LI Li, XUE Tao, LIU Chuanmin
2019, 46(3):  924-930.  doi:10.16431/j.cnki.1671-7236.2019.03.034
Abstract ( 168 )   PDF (2010KB) ( 121 )  
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This study was conducted to investigate the inhibitory effect of NO· on the replication of porcine circovirus type 2 (PCV2).NO was generated from SNP or SNP in the presence of ascorbate (VC).SNP+VC generates mainly NO· in culture medium while NO+ was the major product of SNP alone.PK-15 cells were pretreated with SNP,VC,SNP+VC or control drug (NAP) for 6 h,respectively,then infected with PCV2 (1 MOI) and incubated with the above mentioned drugs for additional 72 h.After incubation,culture medium and cells were collected.IFA,Western blotting,TCID50 and QPCR assay methods were performed to determine the viral titers.The results showed that the highest concentration of SNP,VC and SNP+VC in treated cells were 500,62.5 and 31.25 μmol/L,respectively.The production of NO in 60 μmol/L SNP or 30 μmol/L SNP+30 μmol/L VC treated cells were higher than that in untreated cells and NAP treated cells (P<0.01).Virus titers and PCV2 DNA copies from 30 μmol/L SNP+30 μmol/L VC treated group were significantly lower than that from infected control (P<0.01).In addition,The expression of viral Cap protein from the sample treated with 30 μmol/L SNP+30 μmol/L VC also obviously decreased in relation to those from infected control samples.However,almost no antiviral activities of SNP or VC alone were indicated during PCV2 infection.In conclusion,inhibition of NO on PCV2 replication was mediated by NO·,rather than NO+.

Isolation,Identification and Drug Resistance Analysis of Avian Salmonella in Central China
WANG Dixuan, GAO Dongyang, ZHANG Tianzi, LIU Wei, ZHOU Zutao, XU Xiaojuan, CAI Xuwang
2019, 46(3):  931-939.  doi:10.16431/j.cnki.1671-7236.2019.03.035
Abstract ( 176 )   PDF (2203KB) ( 205 )  
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In order to explore the dominant serovars and antimicrobial resistance of avian Salmonella spp.in Central China,a total of 3 724 issue samples were collected from diseased chickens,dead embryos or weak chick in 22 large-scale farms in Hubei,Henan and Hunan,etc.The isolates and their serotypes were identified by PCR and serotype assay.Kirby-Bauer method was used to analyze the drug resistance of the isolates.The results showed that 124 Salmonella were identified from 3 724 samples,among those 79 strains were Salmonella Enteritidis of D serogroup (63.71%,79/124),34 strains were Salmonella Pullorum of D serogroup (27.42%,34/124),8 strains were Salmonella Typhimurium of B serogroup (6.45%,8/124),whereas 3 strains were failed to be determined.The O antigen of 79 Salmonella Enteritidis and 34 Salmonella Pullorum strains were identified O9 antigen,that of 8 Salmonella Typhimurium strains were O4 antigen.While the H antigen of Salmonella Enteritidis and Salmonella Pullorum strains were identified Hg,m,that of Salmonella Typhimurium strains were Hi.The results of antimicrobial susceptibility test showed that the resistance rate of isolates to nalidixic acid,ampicillin,tetracycline and doxycycline were 95.97% (119/124),91.94% (114/124),57.26% (71/124) and 70.16% (87/124),respectively.The resistance rate to Timethoprim/sulfamethoxazole and erythromycin were 25.81% (32/124) and 12.10% (15/124).The resistance rate to chloramphenicol,gentamicin,cefotaxime and kanamycin were 6.45% (8/124),1.61% (2/124),1.61% (2/124) and 0.81% (1/124),respectively,whereas all isolates were completely sensitive to levofloxacin,amikacin and polymyxin B.Altogether,99.19% (123/124) of these isolates were resistant to at least one drug,and 87.10% (108/124) of isolates showed multiple drug resistance.This study provided the basic data for the diagnosis and prevention of Salmonella in poultry farms in Central China.

Extraction,Purification,Acute Toxicity and Antioxidant Activity of Total Flavonoids from Seeds of Ammopiptanthus
JIN Ye, LIANG Pengfei, FANG Mei, ZHANG Jianan, JIA Ning, DENG Kang, HUANG Weikuan
2019, 46(3):  940-948.  doi:10.16431/j.cnki.1671-7236.2019.03.036
Abstract ( 218 )   PDF (931KB) ( 163 )  
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This experiment was conducted to explore the purification and the acute toxicity and the antioxidant activity of total flavonoids from the seeds of Ammopiptanthus.The total flavonoids from the seeds of Ammopiptanthus was extracted using ultrasonic-assisted extraction method.The content of total flavonoids from the seeds of Ammopiptanthus was measured with AlCl3-CH4O coloration method.The acute toxicity test of total flavonoids was evaluated in mice.The Fenton system of hydroxyl radicals (·OH) produced in vitro and the superoxide radicals (O2-) produced by pyrogallol autoxidation were used to determine the scavenging effect of total flavonoids on radicals.Besides,the effects of total flavonoids on serum malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in aging model mice were measured.The results showed that the precision (RSD=0.78%),reproducibility (RSD=0.52%),stability (RSD=0.65%) and recovery rate (99.140%) of the method for determination of total flavonoids from the seeds of Ammopiptanthus were in the range of error,and the contain of total flavonoids was 86.780%.The total flavonoids from the seeds of Ammopiptanthus belonged to a non-toxic medicine.The total flavonoids from the seeds of Ammopiptanthus had good scavenging ability to ·OH and O2-.In the group of 1 250 μg/mL,there were the best scavenging effects of ·OH and O2-,and the clearance rates reached 96.45% and 50.39%,respectively.The total flavonoids could extremely significantly increase the activity of SOD in the serum compared with aging model mice (P<0.01).In the group of 150 mg/(kg·d) total flavonoids,there was the most significant increase of the activity of SOD in the serum,and the activity reached 102.42 U/mL.Furthermore,the total flavonoids could extremely significantly reduce the content of MDA in the serum compared with aging model mice (P<0.01).In the group of 150 mg/(kg·d) total flavonoids,there was the most significant reduction in the content of MDA in the serum.The results suggested that the total flavonoids from the seeds of Ammopiptanthus had good antioxidant activity and scavenging ability on radicals and repair the immune function of aging model mice.

Clinical Veterinary Medicine
Isolation and Identification of Etiological Bacteria of Subcutaneous Abscess in Goats
ZHANG Siqi, DAO Xiaofang, LI Yao, MA Yuan, YANG Falong
2019, 46(3):  949-956.  doi:10.16431/j.cnki.1671-7236.2019.03.037
Abstract ( 202 )   PDF (2214KB) ( 139 )  
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To identify the main pathogens of goat subcutaneous abscess,a total of 19 abscess samples were collected from three goat farms in Lezhi county,Jintang county and Pengxi county in Sichuan province.The pathogens were detected and identified by bacterial isolation,biochemical test and specific PCR methods,and the drug sensitivity of the pathogens isolated was determined.The results showed that a total of 19 strains of 3 pathogens were isolated by PCR amplification method,and the isolation rate of Corynebacterium pseudotuberculos,Staphylococcus aureus and Arcanobacterium pyogenes was 57.9%(11/19),26.3% (5/19) and 15.8% (3/19),respectively.A total of 17 strains of 3 pathogens were isolated by isolation and identification method,in which 11 strains of Gram-positive Brevibacterium were identified as Corynebacterium pseudotuberculosis whose isolation rate was 57.9%(11/19),4 strains of Gram-positive cocci were identified as Staphylococcus aureus,and isolation rate was 21.1% (4/19),while 2 strains of Gram-positive bacilli were identified as Arcanobacterium pyogenes whose isolation rate was 10.5% (2/19).PCR was more sensitive than bacterial isolation.Three pathogens were sensitive to many drugs such as amikacin and florfenicol,etc.The results of this study provided a useful basis for further analysis of its biological characteristics and the development of comprehensive control measures for subcutaneous abscess in these areas.